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. 2015 Oct 22:15:762.
doi: 10.1186/s12885-015-1718-7.

Regulation of cellular sphingosine-1-phosphate by sphingosine kinase 1 and sphingosine-1-phopshate lyase determines chemotherapy resistance in gastroesophageal cancer

Kasia Matula  1 , Elaina Collie-Duguid  1 , Graeme Murray  1   2 , Khyati Parikh  1 , Heike Grabsch  3 , Patrick Tan  4 , Salina Lalwani  1 , Roberta Garau  1 , Yuhan Ong  1 , Gillian Bain  1   5 , Asa-Dahle Smith  1   6 , Gordon Urquhart  1   5 , Jacek Bielawski  7 , Michael Finnegan  1 , Russell Petty  8
Affiliations

Regulation of cellular sphingosine-1-phosphate by sphingosine kinase 1 and sphingosine-1-phopshate lyase determines chemotherapy resistance in gastroesophageal cancer

Kasia Matula et al. BMC Cancer. .

Abstract

Background: Resistance to chemotherapy is common in gastroesophageal cancer. Mechanisms of resistance are incompletely characterised and there are no predictive biomarkers in clinical practice for cytotoxic drugs. We used new cell line models to characterise novel chemotherapy resistance mechanisms and validated them in tumour specimens to identify new targets and biomarkers for gastroesophageal cancer.

Methods: Cell lines were selected for resistance to oxaliplatin, cisplatin and docetaxel and gene expression examined using Affymetrix Exon 1.0 ST arrays. Leads were validated by qRT-PCR and HPLC of tumour metabolites. Protein expression and pharmacological inhibition of lead target SPHK1 was evaluated in independent cell lines, and by immunohistochemistry in gastroesophageal cancer patients.

Results: Genes with differential expression in drug resistant cell lines compared to the parental cell line they were derived from, were identified for each drug resistant cell line. Biological pathway analysis of these gene lists, identified over-represented pathways, and only 3 pathways - lysosome, sphingolipid metabolism and p53 signalling- were identified as over-represented in these lists for all three cytotoxic drugs investigated. The majority of genes differentially expressed in chemoresistant cell lines from these pathways, were involved in metabolism of glycosphingolipids and sphingolipids in lysosomal compartments suggesting that sphingolipids might be important mediators of cytotoxic drug resistance in gastroeosphageal cancers . On further investigation, we found that drug resistance (IC50) was correlated with increased sphingosine kinase 1(SPHK1) mRNA and also with decreased sphingosine-1-phosphate lysase 1(SGPL1) mRNA. SPHK1 and SGPL1 gene expression were inversely correlated. SPHK1:SGPL1 ratio correlated with increased cellular sphingosine-1-phosphate (S1P), and S1P correlated with drug resistance (IC50). High SPHK1 protein correlated with resistance to cisplatin (IC50) in an independent gastric cancer cell line panel and with survival of patients treated with chemotherapy prior to surgery but not in patients treated with surgery alone. Safingol a SPHK1 inhibitor, was cytotoxic as a single agent and acted synergistically with cisplatin in gastric cancer cell lines.

Conclusion: Agents that inhibit SPHK1 or S1P could overcome cytotoxic drug resistance in gastroesophageal cancer. There are several agents in early phase human trials including Safingol that could be combined with chemotherapy or used in patients progressing after chemotherapy.

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Figures

Fig. 1
Fig. 1
Development and characterisation by gene expression profiling of cytotoxic drug resistant gastroesophageal cancer cell lines. a Drug resistant cell lines used in this study (see also Additional file 1). b Principle component analysis of drug resistant cell lines using 16939 genes expressed in at least 1 cell line (threshold of expression ≥ 20th percentile) with 3 independent replicates per cell line from three different passages using Affymetrix Exon 1.0ST microarrays (see also Additional File 1: Additional information 2). c Only 3 pathways, namely the lysosome, sphingolipid metabolism and p53 signalling were identified as over-represented in gene set enrichment analysis of genes significantly differentially expressed for all 3 cytotoxic drugs compared to sensitive parental lines and in each case they were also identified in at least 2 cell lineages (DAVID v6.7 for biological pathway mapping and gene set enrichment analysis (EASE score, modified Fisher exact p < 0.05 [16]), Paired t-test with Benjamini and Hochberg correction for multiple testing (corrected P <0.05) to derive the differentially expressed gene set, green = over represented red = not over-represented. See also Additional file 1: Additional information 7
Fig. 2
Fig. 2
SPHK1 and SGPL1 expression in cytotoxic drug resistant gastroesophageal cancer cell lines. a All drug resistant cell lines showed increased SPHK1 mRNA expression relative to parental wild type line, and 4 out of 6 also showed decreased SGPL1 mRNA. Data shown is mean (+/- SEM) from 3 independent replicates per cell line from three different passages using Affymetrix Exon 1.0ST microarrays and validated by qRT-PCR (see Additional file 1: Additional information 2 and 3). *** p < 0.01 ** p < 0.05 (Student’s t-test). b Inverse correlation between SPHK1 and SGPL1 mRNA levels (R = -0.740, p = 0.022). Data shown is mean for all drug resistant cell lines and parental wild type lines, 3 independent replicates per cell line from three different passages measured by qRT-PCR (see also Additional file 1: Additional information 5). c Hypothesis of increased SPHK1 and decreased SGPL1 leading to increased S1P in gastro-oeosphageal cancer, promoting cell survival and hence cytotoxic drug resistance
Fig. 3
Fig. 3
Relationship between SPHK1 and SGPL1 expression and S1P and cisplatin resistance in gastroesophageal cancer cell lines. a In drug resistant cell lines that demonstrate increased SPHK1 together with decreased SGPL1 (AGSCIS5, AGSDOC6,OE33OX4, OE33CIS4), the fold change in the ratio of SPHK1:SGPL1 mRNA correlates with observed increase in cellular S1P in drug resistant cell lines relative to the respective parental cell lines (R = 0.981, p = 0.020). Data shown is mean for 3 independent replicates per cell line from three different passages measured by qRT-PCR (see also Additional file 1: Additional information 5). b In the drug resistant cell lines (Fig. 1a), cellular S1P correlates with IC50 to cisplatin, oxaliplatin and docetaxel, respectively (R = 0.690, p = 0.040). IC50 data determined by MTT assay with each data point in each cell line measured in triplicate with 3 independent replicate experiments. S1P measured using high performance liquid chromatography-tandem mass spectrometry as described in the text, mean value from duplicate assays
Fig. 4
Fig. 4
Relationship between SPHK1 protein expression and cisplatin resistance in gastroesophageal cancer cell lines. a SPHK1 protein expression determined by semi-quantitative immunohistochemistry Q-score(see text) correlates with SPHK1 mRNA expression in drug resistant gastroesophageal cell lines(mean for all drug resistant cell lines and parental wild type lines, 3 independent replicates per cell line from three different passages measured by qRT-PCR (see also Additional file 1: Additional information 5). (R = 0.70, p = 0.022). b SPHK1 protein expression determined by semi-quantitative immunohistochemistry Q-score(see text) with IC50 for cisplatin in an independent panel of gastric cancer cell lines. (R = 0.532, p = 0.013)
Fig. 5
Fig. 5
SPHK1 expression in gastroesophageal cancer patients. a SPHK1 Immunohistochemistry. Representative examples (x400) of strong, moderate and weak tumour SPHK1 staining and proportions of tumours in each category. b SPHK1 immunohistochemistry and overall survival of oesophago-gastric cancer patients treated with either surgery alone or with neoadjuvant chemotherapy prior to surgical resection, grouped negative or weak SPHK1 staining(blue line) versus moderate or strong SPHK1 staining (red line) (Kaplan- Meier survival curve, log rank test)
Fig. 6
Fig. 6
Synergistic effects of cisplatin and safingol in gastroesophageal cancer cell lines. a AGSCIS5 cisplatin resistant gastric cancer cell line and b N87 gastric cancer cell line. Cisplatin: Safingol ratio is constant in the combination experiments, and each data point has 6 replicates. Mean growth from three independent experiments shown, relative cell survival ((MTT OD value for cells treated as indicated /MTT OD value for untreated control)*100, ±SEM). Tables show combination index for cisplatin and safingol at different ICs for each cell line and show synergy between the treatments across different doses
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References

    1. Kunz PL, Gubens M, Fisher GA, Ford JM, Lichtensztajn DY, Clarke CA. Long-term survivors of gastric cancer: a California population-based study. J Clin Oncol. 2012;30(28):3507–3515. doi: 10.1200/JCO.2011.35.8028. - DOI - PubMed
    1. Cunningham D, Starling N, Rao S, Iveson T, Nicolson M, Coxon F, Middleton G, Daniel F, Oates J, Norman AR. Capecitabine and Oxaliplatin for advanced esophagogastric cancer. N Engl J Med. 2008;358(1):36–46. doi: 10.1056/NEJMoa073149. - DOI - PubMed
    1. Cunningham D, Allum W, Stenning S, Thompson J, VandeVelde CH, Nicolson M, Scarffe JH, Lofts F, Falk S, Iveson T, Smith D, Langley R, Verma M, Weeden S, Chua Y, theMAGICTrialParticipants Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer. N Engl J Med. 2006;355(1):11–20. doi: 10.1056/NEJMoa055531. - DOI - PubMed
    1. Wagner AD, Grothe W, Haerting J, Kleber G, Grothey A, Fleig WE. Chemotherapy in advanced gastric cancer: a systematic review and meta-analysis based on aggregate data. J Clin Oncol. 2006;24(18):2903–2909. doi: 10.1200/JCO.2005.05.0245. - DOI - PubMed
    1. Van Cutsem E, Moiseyenko VM, Tjulandin S, Majlis A, Constenla M, Boni C, Rodrigues A, Fodor M, Chao Y, Voznyi E, Risse M, Ajani JA. Phase III study of docetaxel and cisplatin plus fluorouracil compared with cisplatin and fluorouracil as first-line therapy for advanced gastric cancer: a report of the V325 study group. J Clin Oncol. 2006;24(31):4991–4997. doi: 10.1200/JCO.2006.06.8429. - DOI - PubMed

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